Mu opioid receptor agonist analogs of the endomorphins

ABSTRACT

The invention relates to cyclic peptide agonists that bind to the mu (morphine) opioid receptor and their use in the treatment of acute and/or chronic pain. Embodiments of the invention are directed to cyclic pentapeptide and hexapeptide analogs of endomorphin that have (i) a carboxy-terminal extension with an amidated hydrophilic amino acid and (ii) a substitution in amino acid position 2. These peptide analogs exhibit decreased tolerance relative to morphine, increased solubility compared to similar tetrapeptide analogs, while maintaining favorable or improved therapeutic ratios of analgesia to side effects.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/477,423, filed on May 22, 2012, which is a continuation-in-part ofPCT/US2011/43306, filed on Jul. 8, 2011, which claims the benefit ofU.S. Provisional Application Ser. No. 61/363,039, filed on Jul. 9, 2010,each of which is incorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

A portion of the work described herein was supported by a Senior CareerResearch Scientist Award and Competitive Merit Review Program fundinggrant from the Department of Veteran Affairs to James E. Zadina. TheUnited States government has certain rights in this invention.

FIELD OF THE INVENTION AND INCORPORATION OF SEQUENCE LISTING

The present invention relates to peptide agonists that bind to the mu(morphine) opioid receptor and their use in the treatment of acute andchronic pain. The biological sequence information in this application isincluded in an ASCII text file having the file name “TU386CIPSEQ.txt”,created on Aug. 24, 2012, and having a file size of 3,011 bytes, whichis incorporated herein by reference.

BACKGROUND OF THE INVENTION

Activation of the mu opioid receptor is among the most effective meansof alleviating a wide range of pain conditions. Of the recently clonedopioid receptors e.g., mu (3,20,21), delta (6,9), and kappa (12-14), thevast majority of clinically used opioids act at the mu receptor. Asillustrated in genetically altered “knock-out” mice, the absence of themu receptor eliminates the analgesic effects of morphine (8),illustrating its central role in opioid-induced pain relief. The uniqueeffectiveness of mu agonists can be attributed to several factors,including their presence in numerous regions of the nervous system thatregulate pain processing and activation of multiple mechanisms thatlimit pain transmission (e g, inhibiting release of excitatorytransmitters from the peripheral nervous system and decreasing cellularexcitability in the central nervous system).

Limitations on the use of opioids result from negative side effects,including abuse liability, respiratory depression, and cognitive andmotor impairment. Major efforts to develop compounds that maintainanalgesic properties while reducing the negative side effects have metwith limited success. This is evident from the recent epidemic ofprescription drug abuse. Numerous attempts at targeting alternativemechanisms of pain relief to avoid these side effects have generallybeen met with similar problems, i.e., a profile of adverse effects thatare different from opioids, but often sufficiently serious to warrantremoval from the market (e.g., COX inhibitors) or lack of approval toenter the market (e.g., TRP receptor antagonists). Over 100 millionpatients annually in the United States experience acute or chronic painand frequently do not achieve adequate relief from existing drugs due tolimited efficacy or excessive side effects.

Elderly patients tend to show greater sensitivity to severe pain andrecent guidelines of the American Geriatric Society suggest greater useof opioids and reduction of non-steroidal anti-inflammatory drugs(NSAIDs) (10). Impairment of motor and cognitive function can be moredebilitating in the elderly than in younger patients, particularly dueto increased risk of fractures (7). Opioids with reduced motor andcognitive impairment are therefore a growing unmet need.

Natural endogenous peptides from bovine and human brain that are highlyselective for the mu opioid receptor relative to the delta or kappareceptor have been described (23 and U.S. Pat. No. 6,303,578 which isincorporated herein by reference in its entirety). These peptides arepotent analgesics and have shown promise of reduced abuse liability (22)and respiratory depression (4,5), as measured in rodent studies. Thelimited metabolic stability of the natural peptides led to thedevelopment of cyclized, D-amino acid-containing tetrapeptide analogs ofthe endomorphins (U.S. Pat. No. 5,885,958 which is incorporated hereinby reference in its entirety) of sufficient metabolic stability toproduce potent analgesia in rodents after peripheral administration. Alead compound from this group reportedly was 3-fold more potent thanmorphine in alleviating neuropathic pain and showed reduced rewardingproperties in animal models that are correlated with abuse potential.While these results are promising, the development of additionalcompounds showing equal or better properties is desirable. The instantinvention addresses this need by providing peptide analogs havingunexpectedly better solubility and side-effect profiles than thepreviously described materials.

SUMMARY OF THE INVENTION

An embodiment of the instant invention is directed to pentapeptide andhexapeptide analogs of endomorphins that differ from the previouslydescribed tetrapeptide analogs by having (i) a carboxy-terminalextension with an amidated hydrophilic amino acid, (ii) a substitutionin amino acid position 2; or (iii) a combination of (i) and (ii). Thepentapeptide and hexapeptide analogs of the present invention exhibitincreased solubility relative to the tetrapeptides while maintainingfavorable therapeutic ratios of analgesia-to-side effects.

The compounds of the present invention are cyclic peptides that act asmu opioid receptor agonists with high affinity. These compounds providerelief of acute pain, chronic pain, or both, and comprise or consist ofcompounds of Formula I:

(I) H-Tyr-cyclo[X₁-X₂-X₃-X₄]-X₅. X₁ and X₄ each independently is anacidic amino acid (i.e., an amino acid comprising a carboxylicacid-substituted side-chain) or a basic amino acid (i.e., an amino acidcomprising an amino-substituted side-chain), with the proviso that if X₁is an acidic amino acid (e.g., D-Asp or D-Glu), then X₄ is a basic aminoacid (e.g., Lys, Orn, Dpr, or Dab), and vice versa. Preferably, X₁ isD-Asp, D-Glu, D-Lys, D-Orn, D-Dpr or D-Dab; while X₄ preferably is Asp,Glu, Lys, Orn, Dpr or Dab. X₂ and X₃ each independently is an aromaticamino acid (i.e., an amino acid comprising an aromatic group in the sidechain thereof). For example, X₂ preferably is Trp, Phe, or N-alkyl-Phe,where the alkyl group preferably comprises 1 to about 6 carbon atoms,i.e., a (C₁ to C₆) alkyl group. X₃ preferably is Phe, D-Phe, or p-Y-Phewhere Y is NO₂, F, Cl, or Br. X₅ is selected from the group consistingof —NHR, Ala-NHR, Arg-NHR, Asn-NHR, Asp-NHR, Cys-NHR, Glu-NHR, Gln-NHR,Gly-NHR, His-NHR, Ile-NHR, Leu-NHR, Met-NHR, Orn-NHR, Phe-NHR, Pro-NHR,Ser-NHR, Thr-NHR, Trp-NHR, Tyr-NHR, and Val-NHR; where R is H or analkyl group (e.g. a (C₁ to C₁₀) alkyl group such as methyl, ethyl,propyl, isopropyl, butyl, isobutyl, pentyl, isopentyl, hexyl, isohexyl,heptyl, or isoheptyl). The peptide of Formula I is cyclic (shown as“c[X₁-X₂-X₃-X₄]” or “cyclo[X₁-X₂-X₃-X₄]” in the formulas describedherein) by virtue of an amide linkage between the carboxylic acid andamino substituents of the side chains of amino acid residues X₁ and X₄.For example, the linkage can be an amide bond formed between the sidechain amino group of the D-Lys, D-Orn, D-Dpr, D-Dab, Lys, Orn, Dpr, orDab with the side chain carboxyl group of D-Asp, D-Glu, Asp, or Glu.

In one embodiment of the invention directed to a peptide of Formula I,X₅ is NHR, R is H, and X₅ can be —NH₂ (i.e., the peptide is an amidatedpentapeptide), or Ala-NH₂, Arg-NH₂, Asn-NH₂, Asp-NH₂, Cys-NH₂, Glu-NH₂,Gln-NH₂, Gly-NH₂, His-NH₂, Ile-NH₂, Leu-NH₂, Met-NH₂, Orn-NH₂, Phe-NH₂,Pro-NH₂, Ser-NH₂, Thr-NH₂, Trp-NH₂, Tyr-NH₂, or Val-NH₂, (i.e., thepeptide is an amidated hexapeptide). In one particular embodiment, X₅ isNH₂. In other particular embodiments, X₅ is Ala-NH₂, Arg-NH₂, Asn-NH₂,Asp-NH₂, Cys-NH₂, Glu-NH₂, Gln-NH₂, Gly-NH₂, His-NH₂, Ile-NH₂, Leu-NH₂,Met-NH₂, Orn-NH₂, Phe-NH₂, Pro-NH₂, Ser-NH₂, Thr-NH₂, Trp-NH₂, Tyr-NH₂,or Val-NH₂.

Another embodiment of the invention is directed to a peptide of FormulaI, wherein X₁ is D-Asp, D-Glu, D-Lys, or D-Orn; and X₄ is Asp, Glu, Lys,or Orn.

Another embodiment of the invention is directed to a compound of FormulaI, wherein X₅ is NHR and R is a (C₁ to C₁₀) alkyl.

Another embodiment of the invention is directed to a peptide of FormulaI, wherein the aromatic amino acid of X₂ is Trp, Phe, or N-alkyl-Phe,and the alkyl group of N-alkyl-Phe is a (C₁ to C₆) alkyl. In oneparticular embodiment, X₂ is N-methyl-Phe (N-Me-Phe).

Another embodiment of the invention is directed to a peptide of FormulaI, wherein the aromatic amino acid residue of either X₂ or X₃ is Phe,D-Phe, Trp, D-Trp, D-Tyr, N-alkyl-Phe, and the alkyl group ofN-alkyl-Phe is (C₁ to C₁₀) alkyl or p-Y-Phe, wherein Y is NO₂, F, Cl, orBr.

Another embodiment of the invention is directed to a peptide of FormulaI, wherein the aromatic amino acid of X₃ is Phe, D-Phe, or p-Y-Phe,wherein Y is NO₂, F, Cl, or Br. In one particular embodiment, X₃ isp-Cl-Phe.

Another embodiment of the invention is directed to a peptide of FormulaI selected from the group consisting of Tyr-c[D-Lys-Trp-Phe-Glu]-NH₂(SEQ ID NO:1); Tyr-c[D-Glu-Phe-Phe-Lys]-NH₂ (SEQ ID NO:2);Tyr-c[D-Lys-Trp-Phe-Glu]-Gly-NH₂ (SEQ ID NO:3);Tyr-c[D-Glu-Phe-Phe-Lys]-Gly-NH₂ (SEQ ID NO:4);Tyr-c[D-Lys-Trp-Phe-Asp]-NH₂ (SEQ ID NO:5);Tyr-c[D-Glu-N-Me-Phe-Phe-Lys]-NH₂ (SEQ ID NO:6); andTyr-c[D-Orn-Phe-p-Cl-Phe-Asp]-Val-NH₂ (SEQ ID NO:7).

Another aspect of the invention is directed to a pharmaceuticalcomposition comprising a peptide of Formula I and a pharmaceuticallyacceptable carrier (e.g., a diluent or excipient).

Yet another aspect of the invention is directed to the use of a peptideof Formula I in a method of treating a patient having a condition thatresponds to an opioid, or a condition for which opioid treatment isstandard in the art. Such a method comprises or consists ofadministering to the patient an effective amount of a peptide of FormulaI of the invention. Particular embodiments of this method can befollowed for the purpose of providing at least one effect selected from(i) analgesia (pain relief), (ii) relief from a gastrointestinaldisorder such as diarrhea, (iii) therapy for an opioid drug dependence,and (iv) treatment of any condition for which an opioid is indicated. Insome embodiments the peptides of Formula I can be used to treat acute orchronic pain. Uses for the peptides of Formula I also include, but arenot be limited to, use as antimigraine agents, immunomodulatory agents,immunosuppressive agents or antiarthritic agents. Certain embodiments ofthe methods of the present invention, such as treatment of pain oropioid drug dependence, are directed to patients having a history ofopioid substance abuse. In certain embodiments of the present methods,the peptide is administered parenterally (e.g., intravenous). Thisinvention also relates to a peptide of Formula I for use in one of saidmethods of treatment.

Another aspect of the invention is directed to a method of activating orregulating a mu-opioid receptor by contacting the mu-opioid receptorwith a compound of the invention, as well as the use of the peptide ofFormula I in such a treatment.

Another aspect of the invention is directed to a method of measuring thequantity of a mu opioid receptor in a sample using a peptide of FormulaI. This method can comprise or consist of the following steps: (i)contacting a sample suspected of containing a mu opioid receptor with apeptide of Formula I to form a compound-receptor complex, (ii) detectingthe complex, and (iii) quantifying the amount of complex formed.

Another aspect of the invention is directed to the use of a peptide ofFormula I to perform a competitive assay method of detecting thepresence of a molecule that binds to a mu opioid receptor. This methodcan comprise or consist of the following steps: (i) contacting a samplesuspected of containing a molecule that binds to a mu opioid receptorwith a mu opioid receptor and a peptide of Formula I, wherein thecompound and receptor form a compound-receptor complex; (ii) measuringthe amount of the complex formed in step (i); and (iii) comparing theamount of complex measured in step (ii) with the amount of a complexformed between the mu opioid receptor and the peptide in the absence ofsaid sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows Tyr-c[D-Lys-Trp-Phe-Glu]-NH₂ (SEQ ID NO:1), which isdescribed as “Compound 1” in the following disclosure. The structuraland basic molecular formulae, as well as the molecular weight (MW), areshown for Compound 1.

FIG. 2 shows Tyr-c[D-Glu-Phe-Phe-Lys]-NH₂ (SEQ ID NO:2), which isdescribed as “Compound 2” in the following disclosure. The structuraland basic molecular formulae, as well as the molecular weight (MW), areshown for Compound 2.

FIG. 3 shows Tyr-c[D-Glu-Phe-Phe-Lys]-Gly-NH₂ (SEQ ID NO:4), which isdescribed as “Compound 4” in the following disclosure. The structuraland basic molecular formulae, as well as the molecular weight (MW), areshown for Compound 4.

FIG. 4 shows opioid receptor binding activity for Compound 1. (A) mureceptor binding of “Compound 1” (triangles) or DAMGO (squares). (B)Antagonist activity of Compound 1 against binding of SNC80 to deltareceptor.

FIG. 5 shows effects of compounds on antinociception and respiration.(A) Effects of Compounds 1 and 2 on antinociception as compared withmorphine. **=p<0.01. (B) Effects of Compounds 1 and 2 on respiratoryminute volume (MV) over a 20-minute period as compared to morphine. *p<0.05. *** p<0.001.

FIG. 6 shows the effects of Compound 2 on antinociception and motorimpairment. (A) The effects of Compound 2 (filled triangles) andmorphine sulfate (MS, filled squares) on antinociception were measuredby the tail flick (TF) test. Also, the effects of Compound 2 (opentriangles) and morphine sulfate (open squares) on motor behavior weremeasured. (*=p<0.05). (B) The bar graph shows the ratio of the areaunder the curve (AUC) for percent motor impairment relative to the AUCfor percent antinociception. This ratio is significantly greater(*p<0.05) for morphine than for Compound 2, consistent with greatermotor impairment relative to analgesia for morphine.

FIG. 7 shows the effects of compounds on drug abuse liability. (A) Theeffects of Compound 1 (filled triangles), morphine (filled squares), andvehicle (filled circles) on antinociception were measured by the tailflick (TF) test. * p<0.05. (B) The cumulative doses of either morphineor Compound 1 that were shown to produce maximal antinociception asshown in (A) were tested for the ability to induce conditioned placepreference (CPP). *** p<0.01.

FIG. 8 shows the duration and relative potency of compounds in reversingchronic pain induced by nerve injury (neuropathic pain). (A) Thedecrease in paw pressure required for withdrawal after nerve injurysurgery was reversed by morphine and Compounds 1, 2, and 5 (squares,down triangles, diamonds, and up triangles, respectively). Times atwhich the reversal was significantly above vehicle (p<0.05 to 0.001) areshown in bars at the top. Scores for Compound 1 were also significantlyabove those of morphine from 155 to 215 min (dashed bar). Compound 5showed similar reversal (80 min) relative to morphine, and Compounds 1and 2 showed significantly longer reversal (120 and 260 min,respectively) relative to morphine. (B) Dose-response curves show thatall three analogs are significantly more potent than morphine, asdetermined by the dose required to fully (100%) reverse hyperalgesia(pre-surgical minus post-surgical pressure).

FIG. 9 shows the extent of tolerance produced by intrathecal delivery ofmorphine or Compound 2 for 1 week via an osmotic minipump. Cumulativedose-response curves (four increasing quarter-log doses) were used andresponses expressed as % maximum possible effect (% MPE) in a tail-flicktest were determined before and after implantation of a minipump. Theshift in ED₅₀ after Compound 2 (about 8.5-fold) was significantly lessthan that after morphine (64 fold), consistent with reduced induction oftolerance by the analog. Similar results were observed with Compounds 1and 5.

FIG. 10 shows activation of glia after 1 week of treatment with morphinebut not analogs. Integrated density of GFAP (A) and pp38 (B) staining inmorphine-treated, but not analog-treated rats is significantly increasedrelative to those given vehicle. In addition, the density of stainingafter morphine is significantly greater than that after analogs (*, **,***=p<0.05, 0.01, 0.001, respectively; n=5-7).

DETAILED DESCRIPTION OF THE INVENTION

Peptides of Formula I, which are cyclic pentapeptide and hexapeptideanalogs of endomorphin-1 (Tyr-Pro-Trp-Phe-NH₂, SEQ ID NO:8) andendomorphin-2 (Tyr-Pro-Phe-Phe-NH₂, SEQ ID NO:9) were prepared. In eachcase, the cyclic portion of the peptide is formed from amino acidresidues 2 through 4, while the Tyr residue (residue 1) is attached toresidue 2 as a branch. Non-limiting examples of peptides with thecomposition of Formula I include Compounds 1-7 below, wherein the sidechains of amino acid residues 2 (X₁) and 5 (X₄) in the sequence arelinked by an amide bond between the side-chains thereof. The formulae ofCompounds 1, 2, 3, 4, 5, 6, and 7 are shown in Table 1.

TABLE 1 Compound H-Tyr- X₁- X₂- X₃- X₄- X₅ SE I ID NO: 1 Tyr- c[D-LysTrp Phe Glu] NH₂ (SEQ ID NO: 1) 2 Tyr- c[D-Glu Phe Phe Lys] NH₂(SEQ ID NO: 2) 3 Tyr- c[D-Lys Trp Phe Glu] Gly-NH₂ (SEQ ID NO: 3) 4Tyr- c[D-Glu Phe Phe Lys] Gly-NH₂ (SEQ ID NO: 4) 5 Tyr- c[D-Lys Trp PheAsp] NH₂ (SEQ ID NO: 5) 6 Tyr- c[D-Glu N-Me-Phe Phe Lys] NH₂(SEQ ID NO: 6) 7 Tyr- c[D-Orn Phe p-Cl-Phe Asp] Val-NH₂ (SEQ ID NO: 7)

In some embodiments, the peptides of Formula I includes peptides with anN-alkylated phenylalanine in position 3 (X₂). Alkyl groups suitable inthe peptides of the present invention include (C₁ to C₁₀) alkyl groups,preferably (C₁ to C₆) alkyl groups (e.g., methyl or ethyl). Compound 6illustrates a cyclic analog whose linear primary amino acid sequencecontains an N-methylated phenylalanine in position 3. Other peptides ofthis invention include compounds wherein the amino acid at position 4(X₃) is p-Y-phenylalanine, wherein Y is NO₂, F, Cl or Br, in order toenhance receptor binding and potency. An exemplary peptide (Compound 7),whose linear primary amino acid sequence is provided in SEQ ID NO:7, hasa p-chlorophenylalanine (p-Cl-Phe) in position 4.

Compounds 1 (FIG. 1), 2 (FIG. 2), 5 and 6 are examples of cyclicpentapeptides, and Compounds 3, 4 (FIG. 3) and 7 are examples of cyclichexapeptides of the instant invention.

For reference, the abbreviations for amino acids described hereininclude alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid(Asp), cysteine (Cys), glutamine (Gln), glutamic acid (Glu), glycine(Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys),methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser),threonine (Thr), tryptophan (Trp), tyrosine (Tyr), valine (Val),ornithine (Orn), naphthylalanine (Nal), 2,3-diaminopropionic acid (Dpr),and 2,4-diaminobutyric acid (Dab). The L- or D-enantiomeric forms ofthese and other amino acids can be included in the peptides of FormulaI. Other amino acids, or derivatives or unnatural forms thereof such asthose listed in the 2009/2010 Aldrich Handbook of Fine Chemicals(incorporated herein by reference in its entirety, particularly thosesections therein listing amino acid derivatives and unnatural aminoacids) can be used in preparing compounds of the invention.

In Formula I, X₁ can be, for example, D-Asp, D-Glu, D-Lys, D-Orn, D-Dpror D-Dab, and X₄ can be, for example, Asp, Glu, Lys, Orn, Dpr or Dab. Ingeneral, an amino acid or derivative thereof can be used as X₁ or X₄ ifit contains either an amino group or a carboxyl group in its side chain.In some embodiments, the amino acid used for X₁ can be a D-enantiomericform of such amino acid.

X₂ and X₃ in Formula I are aromatic amino acids. Examples of such aminoacids are unsubstituted or substituted aromatic amino acids selectedfrom the group consisting of phenylalanine, heteroarylalanine,naphthylalanine (Nal), homophenylalanine, histidine, tryptophan,tyrosine, arylglycine, heteroarylglycine, thyroxine, aryl-beta-alanine,and heteroaryl-beta-alanine. Examples of substituted versions of thesearomatic amino acids are disclosed in U.S. Pat. No. 7,629,319, which isherein incorporated by reference in its entirety. As used herein,“aromatic amino acid” refers to an a-amino acid comprising an aromaticgroup (including aromatic hydrocarbon and aromatic heterocyclic groups)in the side-chain thereof.

In some embodiments, X₂ in Formula I can be N-alkyl-Phe, where the alkylgroup comprises 1 to about 6 carbon atoms. Alternatively, the alkylgroup can comprise about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbons, forexample. The alkyl group can be a methyl (i.e., X₂ is N-Me-Phe), ethyl,propyl, isopropyl, butyl, isobutyl, pentyl, isopentyl, hexyl, isohexyl,heptyl, or isoheptyl group, or any other branched form thereof, forexample. By definition, the alkyl group of N-alkyl-Phe is linked to theα-amino group of phenylalanine. This alpha amino group is involved in anamide bond with the X₁ residue in certain peptides of the invention;therefore, the alpha amino group of X₂ (when N-alkyl-Phe) as it existsin such peptides is a tertiary amide.

In some embodiments X₃ in Formula I is para-Y-Phe (p-Y-Phe), where Y isNO₂, F, Cl, or Br, for example. For example, X₃ can be p-Cl-Phe.Alternatively, the NO₂, F, Cl, or Br groups can be linked in the orthoor meta positions of the phenyl ring of Phe. Any aromatic amino acidincorporated in the compounds of the invention such as at X₂ or X₃ canhave the above groups linked thereto in the ortho, meta, or parapositions.

Solubility.

The solubility of the peptides of Formula I (e.g., in saline orphysiologic buffer) typically is enhanced relative to the prior arttetrapeptide analogs of the endomorphins Addition of a hydrophilic aminoacid and amidated C-terminus to the relatively hydrophobic tetrapeptidesequences Tyr-cyclo[D-Lys-Trp-Phe] (SEQ ID NO:10) andTyr-cyclo[D-Lys-Phe-Phe] (SEQ ID NO:11), resulted in an unexpectedlyhigh improvement in solubility while maintaining or improvingfunctionality. For example, Compound 1 was soluble in water, saline and20% PEG/saline at about 43, 21 and 90 mg/mL, respectively, compared toless than about 2 mg/mL for the previously described compounds. Whileincreases in solubility are associated with improved pharmaceuticaldelivery properties, higher solubility is also often associated withreduced functional activity (e.g., receptor binding) that may depend onlipophilicity. Surprisingly however, as described in examples below, thefunctional properties of the compounds of the invention are notdiminished, and indeed are generally improved.

Methods of Preparation of the Peptides of Formula I.

The peptides of Formula I can be prepared by conventional solution phase(2) or solid phase (18) methods with the use of proper protecting groupsand coupling agents; references 2 and 20 are herein incorporated byreference in their entirety. Such methods generally utilize variousprotecting groups on the various amino acid residues of the peptides. Asuitable deprotection method is employed to remove specified or all ofthe protecting groups, including splitting off the resin if solid phasesynthesis is applied. The peptides can be synthesized, for example, asdescribed below.

Peptides of Formula I were synthesized on Rink Amide resin via Fmocchemistry. A t-butyl group was used for Tyr, Glu, Asp side chainprotection and Boc was used for Lys, Orn and Trp side chain protection.All materials were obtained from EMD Biosciences, Inc (San Diego,Calif.). The peptide was assembled on Rink Amide resin by repetitiveremoval of the Fmoc protecting group and coupling of protected aminoacid. HBTU (O-benzotriazole-N,N,N′,N′-tetramethyluroniumhexafluorophosphate; CAS #94790-37-1) and HOBT (N-hydroxybenzotriazole;CAS #2592-95-2) were used as coupling reagents in N,N-dimethylformamide(DMF) and diisopropylethylamine (DIPEA) was used as a base. The resinwas treated with an aqueous cocktail of trifluoroacetic acid andtriisopropylsilane (TFA/TIS/H₂O cocktail) for cleavage and removal ofthe side chain protecting groups. Crude peptide was precipitated withdiethyl ether and collected by filtration.

Cyclization of the linear Fmoc-Tyr-c[X₁-X₂-X₃-X₄]-X₅ precursors: About 1mmol of peptide was dissolved in about 1000 mL DMF and about 2 mmolDIPEA was added to the solution, followed by a solution of HBTU (about1.1 mmol) and HOBT (about 1.1 mmol) in about 100 mL DMF. The reactionmixture was stirred at room temperature overnight. Solvent was removedin vacuo. The resulting solid residue was washed with 5% citric acid,saturated NaCl, saturated NaHCO₃, and water. The final solid was washedwith diethyl ether and dried under high vacuum.

Preparation of Tyr-c[X₁-X₂-X₃-X₄]-X₅ peptides. The solids obtained abovewere dissolved in 20% piperidine/DMF. The mixture was stirred at roomtemperature for about 1 hour. Solvent was removed in vacuo. Residueswere dissolved in 10% aqueous acetonitrile (MeCN/H₂O) and lyophilized.

Purification of the crude lyophilized peptides was performed withreverse phase high performance liquid chromatography (RP-HPLC). The HPLCsystem GOLD 32 KARAT (Beckman) consisting of the programmable solventmodule 126 and the diode array detector module 168 was used in thepurification and the purity control of the peptides. Reverse phase HPLCwas performed using a gradient made from two solvents: (A) 0.1% TFA inwater and (B) 0.1% TFA in acetonitrile. For preparative runs, a VYDAC218TP510 column (250×10 mm; Alltech Associates, Inc.) was used with agradient of 5-20% solvent B in solvent A over a period of 10 min, 20-25%B over a period of 30 minutes, 25-80% B over a period of 1 minute andisocratic elution over 9 minutes at a flow rate of about 4 mL/min,absorptions being measured at both 214 and 280 nm. The same gradient wasused for analytical runs on a VYDAC 218TP54 column (250×4.6 mm) at aflow rate of about 1 mL/min.

Pharmaceutical Preparations.

The instant invention also provides pharmaceutical preparations whichcontain a pharmaceutically effective amount of the peptides in apharmaceutically acceptable carrier (e.g., a diluent, complexing agent,additive, excipient, adjuvant and the like). The peptide can be presentfor example in a salt form, a micro-crystal form, a nano-crystal form, aco-crystal form, a nanoparticle form, a mirocparticle form, or anamphiphilic form. The carrier can be an organic or inorganic carrierthat is suitable for external, enteral or parenteral applications. Thepeptides of the present invention can be compounded, for example, withthe usual non-toxic, pharmaceutically acceptable carriers for tablets,pellets, capsules, liposomes, suppositories, intranasal sprays,solutions, emulsions, suspensions, aerosols, targeted chemical deliverysystems (15), and any other form suitable for use. Non-limiting examplesof carriers that can be used include water, glucose, lactose, gumacacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc,corn starch, keratin, colloidal silica, potato starch, urea and othercarriers suitable for use in manufacturing preparations, in solid,semisolid, liquid or aerosol form. In addition auxiliary, stabilizing,thickening and coloring agents and perfumes can be used.

The present invention also provides pharmaceutical compositions usefulfor treating pain and related conditions, as described herein. Thepharmaceutical compositions comprise at least one peptide of Formula Iin combination with a pharmaceutically acceptable carrier, vehicle, ordiluent, such as an aqueous buffer at a physiologically acceptable pH(e.g., pH 7 to 8.5), a polymer-based nanoparticle vehicle, a liposome,and the like. The pharmaceutical compositions can be delivered in anysuitable dosage form, such as a liquid, gel, solid, cream, or pastedosage form. In one embodiment, the compositions can be adapted to givesustained release of the peptide.

In some embodiments, the pharmaceutical compositions include, but arenot limited to, those forms suitable for oral, rectal, nasal, topical,(including buccal and sublingual), transdermal, vaginal, parenteral(including intramuscular, subcutaneous, and intravenous), spinal(epidural, intrathecal), and central (intracerebroventricular)administration. The compositions can, where appropriate, be convenientlyprovided in discrete dosage units. The pharmaceutical compositions ofthe invention can be prepared by any of the methods well known in thepharmaceutical arts. Some preferred modes of administration includeintravenous (iv), topical, subcutaneous, oral and spinal.

Pharmaceutical formulations suitable for oral administration includecapsules, cachets, or tablets, each containing a predetermined amount ofone or more of the peptides, as a powder or granules. In anotherembodiment, the oral composition is a solution, a suspension, or anemulsion. Alternatively, the peptides can be provided as a bolus,electuary, or paste. Tablets and capsules for oral administration cancontain conventional excipients such as binding agents, fillers,lubricants, disintegrants, colorants, flavoring agents, preservatives,or wetting agents. The tablets can be coated according to methods wellknown in the art, if desired. Oral liquid preparations include, forexample, aqueous or oily suspensions, solutions, emulsions, syrups, orelixirs. Alternatively, the compositions can be provided as a dryproduct for constitution with water or another suitable vehicle beforeuse. Such liquid preparations can contain conventional additives such assuspending agents, emulsifying agents, non-aqueous vehicles (which mayinclude edible oils), preservatives, and the like. The additives,excipients, and the like typically will be included in the compositionsfor oral administration within a range of concentrations suitable fortheir intended use or function in the composition, and which are wellknown in the pharmaceutical formulation art. The peptides of the presentinvention will be included in the compositions within a therapeuticallyuseful and effective concentration range, as determined by routinemethods that are well known in the medical and pharmaceutical arts. Forexample, a typical composition can include one or more of the peptidesat a concentration in the range of at least about 0.01 nanomolar toabout 1 molar, preferably at least about 1 nanomolar to about 100millimolar.

Pharmaceutical compositions for parenteral, spinal, or centraladministration (e.g. by bolus injection or continuous infusion) orinjection into amniotic fluid can be provided in unit dose form inampoules, pre-filled syringes, small volume infusion, or in multi-dosecontainers, and preferably include an added preservative. Thecompositions for parenteral administration can be suspensions,solutions, or emulsions, and can contain excipients such as suspendingagents, stabilizing agents, and dispersing agents. Alternatively, thepeptides can be provided in powder form, obtained by aseptic isolationof sterile solid or by lyophilization from solution, for constitutionwith a suitable vehicle, e.g. sterile, pyrogen-free water, before use.The additives, excipients, and the like typically will be included inthe compositions for parenteral administration within a range ofconcentrations suitable for their intended use or function in thecomposition, and which are well known in the pharmaceutical formulationart. The peptides of the present invention will be included in thecompositions within a therapeutically useful and effective concentrationrange, as determined by routine methods that are well known in themedical and pharmaceutical arts. For example, a typical composition caninclude one or more of the peptides at a concentration in the range ofat least about 0.01 nanomolar to about 100 millimolar, preferably atleast about 1 nanomolar to about 10 millimolar.

Pharmaceutical compositions for topical administration of the peptidesto the epidermis (mucosal or cutaneous surfaces) can be formulated asointments, creams, lotions, gels, or as a transdermal patch. Suchtransdermal patches can contain penetration enhancers such as linalool,carvacrol, thymol, citral, menthol, t-anethole, and the like. Ointmentsand creams can, for example, include an aqueous or oily base with theaddition of suitable thickening agents, gelling agents, colorants, andthe like. Lotions and creams can include an aqueous or oily base andtypically also contain one or more emulsifying agents, stabilizingagents, dispersing agents, suspending agents, thickening agents,coloring agents, and the like. Gels preferably include an aqueouscarrier base and include a gelling agent such as cross-linkedpolyacrylic acid polymer, a derivatized polysaccharide (e.g.,carboxymethyl cellulose), and the like. The additives, excipients, andthe like typically will be included in the compositions for topicaladministration to the epidermis within a range of concentrationssuitable for their intended use or function in the composition, andwhich are well known in the pharmaceutical formulation art. The peptidesof the present invention will be included in the compositions within atherapeutically useful and effective concentration range, as determinedby routine methods that are well known in the medical and pharmaceuticalarts. For example, a typical composition can include one or more of thepeptides at a concentration in the range of at least about 0.01nanomolar to about 1 molar, preferably at least about 1 nanomolar toabout 100 millimolar.

Pharmaceutical compositions suitable for topical administration in themouth (e.g., buccal or sublingual administration) include lozengescomprising the peptide in a flavored base, such as sucrose, acacia, ortragacanth; pastilles comprising the peptide in an inert base such asgelatin and glycerin or sucrose and acacia; and mouthwashes comprisingthe active ingredient in a suitable liquid carrier. The pharmaceuticalcompositions for topical administration in the mouth can includepenetration enhancing agents, if desired. The additives, excipients, andthe like typically will be included in the compositions of topical oraladministration within a range of concentrations suitable for theirintended use or function in the composition, and which are well known inthe pharmaceutical formulation art. The peptides of the presentinvention will be included in the compositions within a therapeuticallyuseful and effective concentration range, as determined by routinemethods that are well known in the medical and pharmaceutical arts. Forexample, a typical composition can include one or more of the peptidesat a concentration in the range of at least about 0.01 nanomolar toabout 1 molar, preferably at least about 1 nanomolar to about 100millimolar.

A pharmaceutical composition suitable for rectal administrationcomprises a peptide of the present invention in combination with a solidor semisolid (e.g., cream or paste) carrier or vehicle. For example,such rectal compositions can be provided as unit dose suppositories.Suitable carriers or vehicles include cocoa butter and other materialscommonly used in the art. The additives, excipients, and the liketypically will be included in the compositions of rectal administrationwithin a range of concentrations suitable for their intended use orfunction in the composition, and which are well known in thepharmaceutical formulation art. The peptides of the present inventionwill be included in the compositions within a therapeutically useful andeffective concentration range, as determined by routine methods that arewell known in the medical and pharmaceutical arts. For example, atypical composition can include one or more of the peptides at aconcentration in the range of at least about 0.01 nanomolar to about 1molar, preferably at least about 1 nanomolar to about 100 millimolar.

According to one embodiment, pharmaceutical compositions of the presentinvention suitable for vaginal administration are provided as pessaries,tampons, creams, gels, pastes, foams, or sprays containing a peptide ofthe invention in combination with carriers as are known in the art.Alternatively, compositions suitable for vaginal administration can bedelivered in a liquid or solid dosage form. The additives, excipients,and the like typically will be included in the compositions of vaginaladministration within a range of concentrations suitable for theirintended use or function in the composition, and which are well known inthe pharmaceutical formulation art. The peptides of the presentinvention will be included in the compositions within a therapeuticallyuseful and effective concentration range, as determined by routinemethods that are well known in the medical and pharmaceutical arts. Forexample, a typical composition can include one or more of the peptidesat a concentration in the range of at least about 0.01 nanomolar toabout 1 molar, preferably at least about 1 nanomolar to about 100millimolar.

Pharmaceutical compositions suitable for intra-nasal administration arealso encompassed by the present invention. Such intra-nasal compositionscomprise a peptide of the invention in a vehicle and suitableadministration device to deliver a liquid spray, dispersible powder, ordrops. Drops may be formulated with an aqueous or non-aqueous base alsocomprising one or more dispersing agents, solubilizing agents, orsuspending agents. Liquid sprays are conveniently delivered from apressurized pack, an insufflator, a nebulizer, or other convenient meansof delivering an aerosol comprising the peptide. Pressurized packscomprise a suitable propellant such as dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, orother suitable gas as is well known in the art. Aerosol dosages can becontrolled by providing a valve to deliver a metered amount of thepeptide. Alternatively, pharmaceutical compositions for administrationby inhalation or insufflation can be provided in the form of a drypowder composition, for example, a powder mix of the peptide and asuitable powder base such as lactose or starch. Such powder compositioncan be provided in unit dosage form, for example, in capsules,cartridges, gelatin packs, or blister packs, from which the powder canbe administered with the aid of an inhalator or insufflator. Theadditives, excipients, and the like typically will be included in thecompositions of intra-nasal administration within a range ofconcentrations suitable for their intended use or function in thecomposition, and which are well known in the pharmaceutical formulationart. The peptides of the present invention will be included in thecompositions within a therapeutically useful and effective concentrationrange, as determined by routine methods that are well known in themedical and pharmaceutical arts. For example, a typical composition caninclude one or more of the peptides at a concentration in the range ofat least about 0.01 nanomolar to about 1 molar, preferably at leastabout 1 nanomolar to about 100 millimolar.

Optionally, the pharmaceutical compositions of the present invention caninclude one or more other therapeutic agent, e.g., as a combinationtherapy. The additional therapeutic agent will be included in thecompositions within a therapeutically useful and effective concentrationrange, as determined by routine methods that are well known in themedical and pharmaceutical arts. The concentration of any particularadditional therapeutic agent may be in the same range as is typical foruse of that agent as a monotherapy, or the concentration may be lowerthan a typical monotherapy concentration if there is a synergy whencombined with a peptide of the present invention.

In another aspect, the present invention provides for the use of thepeptides of Formula I for treatment of pain, treatment of discomfortassociated with gastrointestinal disorders, and treatment of drugdependence. Methods for providing analgesia (alleviating or reducingpain), relief from gastrointestinal disorders such as diarrhea, andtherapy for drug dependence in patients, such as mammals, includinghumans, comprise administering to a patient suffering from one of theaforementioned conditions an effective amount of a peptide of Formula I.Diarrhea may be caused by a number of sources, such as infectiousdisease, cholera, or an effect or side-effect of various drugs ortherapies, including those used for cancer therapy. Preferably, thepeptide is administered parenterally or enterally. The dosage of theeffective amount of the peptides can vary depending upon the age andcondition of each individual patient to be treated. However, suitableunit dosages typically range from about 0.01 to about 100 mg. Forexample, a unit dose can be in the range of about 0.2 mg to about 50 mg.Such a unit dose can be administered more than once a day, e.g., two orthree times a day.

All of the embodiments of the peptides of Formula I can be in the“isolated” state. For example, an “isolated” peptide is one that hasbeen completely or partially purified. In some instances, the isolatedcompound will be part of a greater composition, buffer system or reagentmix. In other circumstances, the isolated peptide may be purified tohomogeneity. A composition may comprise the peptide or compound at alevel of at least about 50, 80, 90, or 95% (on a molar basis or weightbasis) of all the other species that are also present therein. Mixturesof the peptides of Formula I may be used in practicing methods providedby the invention.

Additional embodiments of the current invention are directed towardsmethods of using the peptides of Formula I disclosed herein in medicinalformulations or as therapeutic agents, for example. These methods mayinvolve the use of a single peptide, or multiple peptides in combination(i.e., a mixture). Accordingly, certain embodiments of the invention aredrawn to medicaments comprising the peptides of Formula I, and methodsof manufacturing such medicaments.

As used herein, the terms “reducing,” “inhibiting,” “blocking,”“preventing”, alleviating,” or “relieving” when referring to a compound(e.g., a peptide), mean that the compound brings down the occurrence,severity, size, volume, or associated symptoms of a condition, event, oractivity by at least about 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, 22.5%,25%, 27.5%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%,or 100% compared to how the condition, event, or activity would normallyexist without application of the compound or a composition comprisingthe compound. The terms “increasing,” “elevating,” “enhancing,”“upregulating”, “improving,” or “activating” when referring to acompound mean that the compound increases the occurrence or activity ofa condition, event, or activity by at least about 7.5%, 10%, 12.5%, 15%,17.5%, 20%, 22.5%, 25%, 27.5%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 400%, 500%, 750%, or1000% compared to how the condition, event, or activity would normallyexist without application of the compound or a composition comprisingthe compound.

The following examples are included to demonstrate certain aspects ofthe invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples, which representtechniques known to function well in practicing the invention, can beconsidered to constitute preferred modes for its practice. However,those of skill in the art should, in light of the present disclosure,appreciate that many changes can be made in the specific disclosedembodiments and still obtain a like or similar result without departingfrom the spirit and scope of the invention. The examples are providedfor illustration purposes only and are not intended to be limiting.

Example 1 Binding and Activation of Human Opioid Receptors

The peptides of Formula I showed surprisingly high affinity(subnanomolar) for the human mu opioid receptor with selective bindingrelative to the delta and kappa opioid receptors. The compounds weretested in standard binding assays using ³H-DAMGO (tritiated [D-Ala²,N-Me-Phe⁴, Gly-o1]-enkephalin; CAS #78123-71-4), ³H-DPDPE(CAS#88373-73-3), and ³H-U69593 (CAS#96744-75-1) to label mu, delta andkappa receptors, respectively, in membranes from CHO cells expressinghuman cloned receptors. As shown in Table 2, endomorphin-1 (EM1, SEQ IDNO:8) and endomorphin-2 (EM2, SEQ ID NO:9) are the most selectiveendogenous mu agonists previously reported. Analogs based on thesenatural opioids show greater affinity for the mu receptor, albeit withless selectivity. Tetrapeptide endomorphin analogs described earlier(U.S. Pat. No. 5,885,958; ck1, Tyr-c[D-Lys-Trp-Phe] (SEQ ID NO:10); ck2,Tyr-c[D-Lys-Phe-Phe] (SEQ ID NO:11)) showed the highest affinity of thecompounds tested. Peptides of Formula I, which include a hydrophilicamino acid and amidated carboxy-terminus (Compounds 1, 2, 5) retainedhigh affinity binding, but increased selectivity for the mu receptor.

TABLE 2 Compound binding to opioid receptors. K_(i) (nM) Selectivity MuDelta Kappa Delta/Mu Kappa/Mu Morphine 0.92 242 56 264 61 DAMGO 0.78 589334 754 429 EM1 2.07 1215 >10000 587 >5000 EM2 1.32 5704 >100004328 >5000 ck1 0.32 28 35 90 111 ck2 0.36 3 12 9 33 Compound 1 0.49 132128 267 260 Compound 2 0.73 69 71 94 98 Compound 5 0.43 140 29 328 67

Receptor Activation: GTPγS Functional Assay.

Functional activation of the three opioid receptors was tested instandard assays in which the non-hydrolysable GTP analog, ³⁵S-GTPγS, wasused to quantify activation of cloned human opioid receptors expressedin cell membranes. FIG. 4A shows that Compound 1 is a full efficacyagonist with significantly greater potency than the reference compound,DAMGO. FIG. 4B shows that Compound 1 exhibits unexpected full efficacyas a delta antagonist; i.e., it is able to inhibit the delta activationproduced by an ED₈₀ dose of the reference delta agonist, SNC80 (CAS#156727-74-1). Table 3 shows that all agonists tested are potentactivators of the mu receptor, with EC₅₀ (median effectiveconcentration) values at low-nanomolar to sub-nanomolar concentrations.All compounds were found to be full efficacy (>90%) agonists at the mureceptor. The endomorphins and the compounds of Formula I of theinvention show remarkable selectivity for receptor activation, withdelta activation below 50% at concentrations up to 10 μM, reflectingselectivity >100000. Compounds 1 and 3, however, showed full efficacydelta antagonism; Compound 1 exhibited this antagonism at a relativelylow concentration.

TABLE 3 Opioid receptor activation by compounds. Agonist EC₅₀ (nM)Selectivity Delta Antagonist mu delta kappa delta/mu kappa/mu IC 50efficacy MS^(a) 3.90 1245 2404 319 616 DAMGO 1.98 3641 13094 1839 6613ck1 0.21 138 469.51 658 2236 ck2 0.15 7 206.11 44 1374 EM11.82 >100000 >100000 >50000 >50000 4287 100 EM28.44 >100000 >100000 >10000 >10000 30000 88 Comp. 1 0.15 >100000963.79 >500000 6425 105 93 Comp. 2 0.99 >100000 12114.00 >100000 122362750 51 Comp. 5 0.22 >100000 740.34 >400000 3365 557 100 ^(a)morphinesulfate

Receptor Activation: Beta-Arrestin Recruitment.

Beta-arrestin is an intracellular protein that is recruited to the muopioid receptor following activation by agonists. It has been shown toactivate intracellular signaling pathways that in many cases areindependent of well-known G-protein mediated pathways. It has recentlybeen shown that beta-arrestin knockout mice exhibit altered responses tomorphine, including increased analgesia and decreased side effects suchas tolerance, respiratory depression, and constipation (16). Theseresults indicate that the analgesic and side-effects of morphine areseparable by manipulation of cell signaling processes. These findingsalso provide support for the recent concept known variously as“functional selectivity”, “biased agonism”, “agonist directed signaling”and other descriptions. According to this concept, agonists capable ofproducing a different cascade of signaling at a given receptor couldproduce a different profile of desired and undesired effects relative toother agonists for that receptor. Three of the analogs of this inventionwere tested and showed patterns of beta-arrestin recruitment (rangingfrom high potency with low efficacy to moderate potency with significantefficacy) that were different from each other and from morphine.Together with the differential analgesic/side-effect profiles relativeto morphine described in previous examples, the beta arrestin resultssuggest that these compounds exhibit “functional selectivity”, favoringanalgesia over adverse side-effects.

Beyond the value of high mu agonist selectivity (i.e., exclusion ofpotential side-effects resulting from activation of multiple receptors),delta antagonism is expected to attenuate opioid-induced tolerance,dependence, and reward. As first shown in 1991 (1) and supported innumerous studies since, delta antagonists can reduce morphine-inducedtolerance and dependence, while maintaining or enhancing analgesia.Recent studies (11) have also shown reduced rewarding properties of muagonist/delta antagonists as reflected in the conditioned placepreference (CPP) test described below. The activity of the peptides ofFormula I (e.g., Compound 1) as mu agonists/delta antagonists as well asat mu/delta receptor dimers indicate that the peptides will produceeffective analgesia with reduced tolerance, dependence, and reward (18).

Example 2 Providing Analgesia of Greater Duration, but with ReducedRespiratory Depression, Relative to Morphine After IntravenousAdministration

Respiratory depression is a major safety issue in the use of opioids. Anopioid providing analgesia as effective as that produced by morphine,but with less respiratory depression, would be a major advance for thesafe use of opioid analgesics. Effectiveness after systemicadministration, such as intravenous (i.v.) injection, is unusual forpeptide-based compounds, and would be critical for the clinical utilitythereof. Two peptides (Compounds 1 and 2) were tested for their effectson respiration (minute ventilation) and duration of antinociceptionrelative to morphine. Rats with indwelling jugular catheters were placedin a BUXCO whole body plethysmograph apparatus for determining multiplerespiratory parameters. For 20 minutes following i.v. injection ofvehicle (saline), baseline minute ventilation was determined. Animalswere then injected with morphine or test compound and changes frombaseline were determined for 20 minutes, the period of maximalinhibition of minute ventilation by all compounds. The standardtail-flick (TF) test was used to determine antinociception. A baselinetest was conducted before placing the animal in the BUXCO chamber, atthe end of the 20-minute respiratory test, and at every 20 minutesthereafter until the TF latency returned to below 2× baseline TF.Baseline latencies were 3-4 seconds and a cut-off time (“maximalantinociception”) was set at 9 seconds to avoid tissue damage.

FIG. 5A shows that 10 mg/kg doses of Compounds 1 and 2 producedsignificantly longer antinociception than all other treatments(**=p<0.01) and 5.6 mg/kg doses produced antinociception similar to the10 mg dose of morphine. Despite the greater antinociceptive effect ofCompounds 1 and 2, significantly (* p<0.05) less inhibition ofrespiration was observed in both doses of Compound 1 and in the 5.6mg/kg dose of Compound 2 (FIG. 5B). These results indicate an unexpectedand clearly safer therapeutic profile for the peptides of Formula I overthe current standard opioid analgesic.

Example 3 Providing Analgesia of Greater Duration than Morphine withReduced Impairment of Neuromotor Coordination and Cognitive Function

Neuromotor and cognitive impairment are characteristics of opioids thatare of particular importance in two populations, i.e., military combattroops, where escape from immediate danger can require unimpaired motorand cognitive skills, and the elderly, where these impairments canexacerbate compromised function including impaired balance, which canlead to increased risk of fractures.

Example 3a Neuromotor Coordination

FIG. 6A illustrates that Compound 2 produces significantly greaterantinociception, but significantly reduced motor impairment, relative tomorphine (MS). Both compounds were administered by cumulativeintravenous (i.v.) doses in rats. Increasing quarter-log doses weregiven every 20 minutes, and a tail flick (TF) test (a test of latency toremove the tail from a hot light beam) followed by a rotorod test wereconducted about 15 minutes after each injection. Escalating doses weregiven until each animal showed greater than 90% maximum possible effect(% MPE) on the TF test, determined as: [(latency to TF minus baselinelatency)/(9 sec maximum (cut off) time to avoid tissue damage) minusbaseline)]×100. The animal was then placed on a rod that rotated atspeeds escalating to 13 revolutions per minute (RPM) over 3 minutes, andthe latency to fall from the rod was determined. Only animals thatconsistently remained on the rod for the full 180 seconds duringtraining in the drug-naïve state were tested. % Maximum PossibleInhibition (% MPI) of motor coordination was determined as 100−(latencyto fall/180×100).

The two compounds showed similar onset to maximal antinociception, butCompound 2 produced significantly longer antinociception, as reflectedby TF latencies significantly (*=p<0.05) longer than those of themorphine group at 135 and 155 minutes (FIG. 6A). Despite this greaterantinociception, the motor impairment was significantly less than thatof morphine (FIG. 6B, * p<0.05). The impairment of motor behavior bymorphine was significantly above that of vehicle controls (* p<0.05)while that of Compound 2 was not.

Example 3b Cognitive Impairment

A widely used standard test of cognitive function is the Morris WaterMaze (MWM). During training, rats learn to find a hidden escape platformbased on spatial memory. Average latency to the platform, as well asaverage distance from the platform (a measure unaffected by swim speed),decrease as the task is acquired and provide indices of spatial memory.After 4 days of training, an injection of morphine produced impairmentof spatial memory, as reflected by a significant increase in the latencyto, and average distance from, the platform. By contrast, Compound 2, atdoses that provide equal or greater antinociception than morphine, didnot produce significant impairment. These results indicate an unexpectedand superior therapeutic profile of the peptides of Formula I withregard to cognitive function relative to the current standard opioidanalgesic.

Example 4 Providing Analgesia of Greater Duration, but Reduced Reward,Relative to Morphine

Opioids remain the standard treatment for relief of severe pain, butdiversion of pain medications for non-pain use has become a seriousnational problem (see U.S. Department of Health and Human ServicesSubstance Abuse and Mental Health Services Administration, found atworld wide websiteoas.samhsa.gov/2k9/painRelievers/nonmedicalTrends.pdf). Considerableefforts in academia and industry have focused on “tamper-proof” versionsof opioid medications, but there has been little success in developingopioids that provide highly effective analgesia with minimal abusepotential. The conditioned place preference (CPP) paradigm is a widelyaccepted model for demonstrating rewarding properties of drugs, and allmajor classes of abused drugs produce CPP, including opioids such asmorphine and heroin. Briefly, animals are first allowed, on Day 1, tofreely explore a 3-compartment apparatus consisting of a small “startbox” and two larger compartments that are perceptually distinct (grayvs. black and white stripes in this example). For the next three days,the animals are given an i.v. injection of drug and confined to onecompartment, and vehicle is given in the other. The time at which thedrug or vehicle is given (a.m. or p.m.) is counterbalanced, as is thecompartment in which the drug is given (preferred or non-preferred, asdetermined during the baseline test). This unbiased design allows fordetection of both drug preference and drug aversion. After three days ofconditioning (Days 2, 3 and 4), the animal is allowed free access to allcompartments on Day 5 in the drug-free state and the change in absolutetime and proportion of time spent in the drug-paired compartment aredetermined. A significant increase in the time or proportion of timespent in the drug-paired compartment on the post-conditioning test dayrelative to that on the pre-conditioning baseline test is interpreted asa conditioned place preference, reflective of rewarding properties andpotential abuse liability.

When the cumulative doses of either morphine or Compound 1 that wereshown to produce maximal antinociception (FIG. 7A) were tested for theability to induce CPP (FIG. 7B), morphine produced a significant (***p<0.01) increase in the time spent on the drug side, while Compound 1did not, even though significantly (* p<0.05) greater antinociception(FIG. 7A) was observed with Compound 1 from about 140 to 180 minutesafter its injection. Compounds 2 and 5 also showed no significant CPP atdoses producing antinociception equal to those of morphine that producedCPP. In a complementary paradigm in which rats were provided access tomorphine or EM analogs for self-administration, access to morphine, butnot analogs, resulted in significant self-administration. These findingsare consistent with less abuse liability for the novel analogs relativeto morphine.

Example 5 Alleviation of Chronic Pain

Chronic pain affects a large proportion of the population. One form ofchronic pain, neuropathic pain, is particularly difficult to treat. FIG.8 shows that Compounds 1, 2 and 5 provide unexpectedly potent relief ofneuropathic pain induced by the spared nerve injury (SNI) model in therat. As demonstrated in FIG. 8A, prior to SNI surgery (“pre-surgery”),an average pressure of about 177 g applied to the hindpaw with aRandall-Selitto device was required to elicit a paw withdrawal response.About 7 to 10 days post-surgery, the animals showed hyperalgesia,indicated by a reduction in the average pressure (to about 70 g)required to elicit withdrawal. Drugs were administered as intrathecalcumulative doses chosen to produce full alleviation of the hyperalgesia.Times at which the reversal was significantly (p<0.05 to 0.001) abovevehicle are shown in bars at the top. Compound 5 showed similar reversaltimes (about 80 min), and Compounds 1 and 2 showed significantly longerreversal times (about 120 and 260 min, respectively) relative tomorphine (about 80 min). Scores for Compound 1 were also significantlyabove those of morphine from 155-215 min (dashed bar). Dose-responsecurves (FIG. 8B) showed that all three analogs are significantly morepotent than morphine, as determined by the dose required to fully (100%)reverse the hyperalgesia, i.e., return to the pre-surgical baselineresponse (presurgical minus post surgical pressure). Compounds 1, 2, and5 reversed mechanical hypersensitivity at doses about 80-fold to100-fold lower than morphine (about 0.01 to 0.014 μg compared to about1.14 μg for morphine). On a molar basis, this represents about 180 to240 fold greater potency than morphine against neuropathic pain. Similarresults were observed after other forms of chronic pain includingpost-incisional (post-operative) and inflammatory pain induced byComplete Freund's Adjuvant (CFA). The foregoing examples areillustrative, but not exhaustive, as to the types of acute or chronicpain for which the peptides of Formula I are effective.

Example 6 Reduced Tolerance and Glial Activation Relative to Morphine

A major limiting factor for the usefulness of opioid medications istolerance, which requires increasing doses to maintain an analgesiceffect. Reduction of the potential for tolerance would be a veryimportant advantage for a novel analgesic. In addition, several recentstudies have shown that repeated opioid exposure sometimes leads to“paradoxical” opioid-induced pain. Increased responsiveness to normallynoxious stimuli (hyperalgesia) or normally non-noxious stimuli such astouch (allodynia) have been reported. Explanations for the tolerance andopioid induced hypersensitivity include the possibility that activationof glia, a reflection of an inflammatory response, results in anincreased release of substances that activate or sensitize neuronaltransmission of nociceptive signals. Specifically, enhanced release of“pronociceptive” cytokines and chemokines are thought to mediate theenhanced pain sensitivity sometimes observed after chronic exposure toopioids. In addition, several studies have linked this phenomenon toopioid tolerance based on the concept that increasing doses of opioidsare required to overcome the increased pronociceptive effects of thereleased compounds. Described below are the unexpected findings that:(1) Compounds 1, 2 and 5 produce significantly less tolerance relativethan morphine, and (2) that in direct comparison to morphine, and incontrast to morphine and most clinically used opioids, the analogs donot induce an inflammatory glial activation response after chronicadministration. In addition to their potential value for reducedescalation of doses required during chronic administration, the analogsof Formula I could be ideal for opioid rotation and for a wide range ofsituations where ongoing inflammatory conditions may be exacerbated bytreatment with morphine. This approach would also be superior to use ofan anti-inflammatory agent as an adjuvant to opioid treatment.

Compounds 1, 2 and 5 all showed greater potency, reduced tolerance andreduced glial activation relative to morphine. For simplicity, onlyCompound 2 is shown in comparison to morphine in FIG. 9. The experimentwas designed to model clinical use of opioids by titrating to fullantinociception in each subject, and maintaining steady blood levels, inthis case through use of osmotic minipumps. Doses producing matchedinitial antinociception were determined for morphine and analog byintrathecal injection of the cumulative dosing paradigm described abovefor the rotorod and neuropathic pain models. Doses were increased untileach rat achieved full antinociception (100% MPE). The ED₅₀ for allcompounds in opioid naïve animals was determined and Compound 2 wasfound to be over 20-fold more potent (p<0.001) than morphine (ED₅₀=0.01μg±0.001 compared to 0.253 μg±0.05 for morphine, n=5-7). This translateson a molar basis to about 40-fold greater potency for the analog.Immediately after the first test, ALZET osmotic minipumps (Durect Corp,Cupertino, Calif.) were implanted subcutaneously and connected to theintrathecal catheter. The primed pumps delivered morphine or analog at 2μg/hr or 0.056 μg/hr for about 7 days, respectively. The 2 μg/hrmorphine dose was chosen based on previous studies in which this dosewas shown to produce glial activation in the dorsal horn in a similarparadigm (Tawfik et al., 2005). The dose of analog was chosen using asimilar ratio to the ED₅₀ (about 7× to 8×). A second cumulativedose-response curve was generated on Day 7 after minipump implantationto determine the shift in ED₅₀ as an index of relative tolerance. Asshown in FIG. 9, the ED₅₀ of morphine shifted to 16±3.3 μg (over60-fold) while that of compound 2 shifted only about 8.5 fold to about0.11±0.02 μg. Compounds 1 and 5 showed similar results with potenciesover 20× greater than morphine and shifts less than 20 fold. Theseresults show that EM analogs cause unexpected and significantly lesstolerance than morphine.

As shown in FIG. 10, morphine produced significant glial activation, butfor all 3 analogs, activation was not significantly different fromvehicle and was significantly less than morphine, establishingdifferential glial effects for morphine compared to EM analogs(Compounds, 1, 2, and 5). Rats used in the above tolerance experimentwere perfused after the final behavioral test and analyzed for glialactivation as indicated by (A) GFAP staining for astroglia and (B)phospho-p38, a signaling pathway activated in microglia by morphine.Five sections from each of 5-7 animals/group were analyzed forintegrated density of staining with the IMAGE J program. Morphine, butnone of the analogs, showed significantly greater induction thanvehicle. Values for all analogs were significantly below those ofmorphine (*,**,***=p<0.05, 0.01,0.001, respectively, compared toindicated groups). These data provide evidence that, at doses producingequal or greater antinociception, the analogs produce unexpectedly lessglial activation and this is associated with reduced tolerance.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

REFERENCES

The following references are referred to in this application and areincorporated herein by reference in their entirety:

-   (1) Abdelhamid E. E., Sultana M., Portoghese P. S. and    Takemori A. E. (1991) Selective blockage of delta opioid receptors    prevents the development of morphine tolerance and dependence in    mice. J. Pharmacol. Exp. Ther. 258, 299-303;-   (2) Bodanszky M. (1993) Peptide Chemistry: A Practical Textbook.    Springer-Verlag, New York;-   (3) Chen Y., Mestek A., Liu J., Hurley J. A. and Yu L. (1993)    Molecular cloning and functional expression of a μ-opioid receptor    from rat brain. Mol. Pharmacol. 44, 8-12;-   (4) Czapla M. A., Gozal D., Alea O. A., Beckerman R. C. and    Zadina J. E. (2000) Differential cardiorespiratory effects of    endomorphin 1, endomorphin 2, DAMGO, and morphine. Am. J. Respir.    Crit Care Med 162, 994-999;-   (5) Czapla M. A. and Zadina J. E. (2005) Reduced suppression of    CO₂-induced ventilatory stimulation by endomorphins relative to    morphine. Brain Res. 1059, 159-166;-   (6) Evans C. J., Keith D. E., Jr., Morrison H., Magendzo K. and    Edwards R. H. (1992) Cloning of a delta opioid receptor by    functional expression. Science 258, 1952-1955;-   (7) Gianni W., Ceci M., Bustacchini S, Corsonello A., Abbatecola A.    M., Brancati A. M., Assisi A., Scuteri A., Cipriani L. and    Lattanzio F. (2009) Opioids for the treatment of chronic non-cancer    pain in older people. Drugs Aging 26 Suppl 1, 63-73;-   (8) Kieffer B. L. (1999) Opioids: first lessons from knockout mice.    Trends Pharmacol. Sci 20, 19-26;-   (9) Kieffer B. L., Befort K., Gaveriaux-Ruff C. and    Hirth C. G. (1992) The δ-opioid receptor: isolation of a cDNA by    expression cloning and pharmacological characterization. Proc. Natl.    Acad. Sci U. SA 89, 12048-12052;-   (10) Kuehn B. M. (2009) New pain guideline for older patients: avoid    NSAIDs, consider opioids. JAMA 302, 19;-   (11) Lenard N. R., Daniels D. J., Portoghese P. S. and    Roerig S. C. (2007) Absence of conditioned place preference or    reinstatement with bivalent ligands containing mu-opioid receptor    agonist and delta-opioid receptor antagonist pharmacophores. Eur. J.    Pharmacol. 566, 75-82;-   (12) Meng F., Xie G. X., Thompson R. C., Mansour A., Goldstein A.,    Watson S. J. and Akil H. (1993) Cloning and pharmacological    characterization of a rat K opioid receptor. Proc. Natl. Acad. Sci    U.SA 90, 9954-9958;-   (13) Minami M., Toya T., Katao Y., Maekawa K., Nakamura S., Onogi    T., Kaneko S. and Satoh M. (1993) Cloning and expression of a cDNA    for the rat κ-opioid receptor. FEBS Lett. 329, 291-295;-   (14) Nishi M., Takeshima H., Fukuda K., Kato S. and Mori K. (1993)    cDNA cloning and pharmacological characterization of an opioid    receptor with high affinities for K-subtype-selective ligands. FEBS    Lett. 330, 77-80;-   (15) Prokai-Tatrai K., Prokai L. and Bodor N. (1996) Brain-targeted    delivery of a leucine-enkephalin analogue by retrometabolic    design. J. Med Chem. 39, 4775-4782;-   (16) Raehal, K M, J K L Walker, and L M Bohn (2005) Morphine Side    Effects in β-Arrestin 2 Knockout Mice. J. Pharmacol. Exp. Ther. 314,    1195-1201-   (17) Rozenfeld R. and Devi L. A. (2010) Receptor heteromerization    and drug discovery. Trends Pharmacol. Sci 31, 124-130;-   (18) Stewart J. M. and Young J. D. (1984) Solid Phase Peptide    Synthesis. Pierce Chemical Company;-   (19) Tawfik V. L., LaCroix-Fralish M. L., Nutile-McMenemy N.,    DeLeo J. A. (2005) Transcriptional and translational regulation of    glial activation by morphine in a rodent model of neuropathic    pain. J. Pharmacol. Exp. Ther. 313, 1239-1247;-   (20) Thompson R. C., Mansour A., Akil H. and Watson S. J. (1993)    Cloning and pharmacological characterization of a rat μ opioid    receptor. Neuron 11, 903-913;-   (21) Wang J. B., Johnson P. S., Persico A. M., Hawkins A. L.,    Griffin C. A. and Uhl G. R. (1994) Human μ opiate receptor. cDNA and    genomic clones, pharmacologic characterization and chromosomal    assignment. FEBS Lett. 338, 217-222;-   (22) Wilson A. M., Soignier R. D., Zadina J. E., Kastin A. J.,    Nores W. L., Olson R. D. and Olson G. A. (2000) Dissociation of    analgesic and rewarding effects of endomorphin-1 in rats. Peptides    21, 1871-1874; and-   (23) Zadina J. E., Hackler L., Ge L. J. and Kastin A. J. (1997) A    potent and selective endogenous agonist for the μ-opiate receptor.    Nature 386, 499-502.

1. A cyclic peptide of Formula I:H-Tyr-cyclo[X₁-X₂-X₃-X₄]-X₅  (I), wherein X₁ and X₄ each independentlyis an acidic amino acid or a basic amino acid; X₂ and X₃ eachindependently is an aromatic amino acid; X₅ is Ala-NHR, Arg-NHR,Asn-NHR, Asp-NHR, Cys-NHR, Glu-NHR, Gln-NHR, Gly-NHR, His-NHR, Ile-NHR,Leu-NHR, Met-NHR, Orn-NHR, Phe-NHR, Pro-NHR, Ser-NHR, Thr-NHR, Trp-NHR,Tyr-NHR, or Val-NHR, wherein R is H or an alkyl group; and there is anamide bond between an amino group and a carboxylic acid group on sidechains of amino acids X₁ and X₄, with the proviso that when X₁ is anacidic amino acid, then X₄ is a basic amino acid; and when X₁ is a basicamino acid, then X₄ is an acidic amino acid.
 2. The peptide of claim 1wherein: (i) X₁ is selected from the group consisting of D-Lys, D-Orn,Lys, and Orn; and X₄ is selected from the group consisting of D-Asp,D-Glu, Asp, and Glu; or (ii) X₁ is selected from the group consisting ofD-Asp, D-Glu, Asp, and Glu; and X₄ is selected from the group consistingof D-Lys, D-Orn, Lys, and Orn.
 3. The peptide of claim 1, wherein: X₂ isselected from the group consisting of Trp, Phe, and N-alkyl-Phe, whereinthe alkyl group of N-alkyl-Phe comprises 1 to about 6 carbon atoms; andX₃ is selected from the group consisting of Phe, D-Phe, and p-Y-Phe,wherein Y is NO₂, F, Cl, or Br.
 4. The peptide of claim 3, wherein X₂ isN-methyl-Phe.
 5. The peptide of claim 3, wherein X₃ is p-Cl-Phe. 6.(canceled)
 7. The peptide of claim 1, wherein R is H and X₅ is Ala-NH₂,Arg-NH₂, Asn-NH₂, Asp-NH₂, Cys-NH₂, Glu-NH₂, Gln-NH₂, Gly-NH₂, His-NH₂,Ile-NH₂, Leu-NH₂, Met-NH₂, Orn-NH₂, Phe-NH₂, Pro-NH₂, Ser-NH₂, Thr-NH₂,Trp-NH₂, Tyr-NH₂, or Val-NH₂.
 8. The peptide of claim 1, wherein thealkyl group is a methyl, ethyl, propyl, isopropyl, butyl, isobutyl,pentyl, isopentyl, hexyl, isohexyl, heptyl, or isoheptyl group.
 9. Thepeptide of claim 1, selected from the group consisting of:Tyr-cyclo[D-Lys-Trp-Phe-Glu]-Gly-NH₂ (SEQ ID NO:3),Tyr-cyclo[D-Glu-Phe-Phe-Lys]-Gly-NH₂ (SEQ ID NO:4), andTyr-cyclo[D-Orn-Phe-p-Cl-Phe-Asp]-Val-NH₂ (SEQ ID NO:7).
 10. Apharmaceutical composition comprising a pharmaceutically acceptablecarrier and the peptide of claim
 1. 11. A method of treating paincomprising administering to a subject an analgesic amount of the peptideof claim
 1. 12. The method of claim 11, wherein the pain is due to agastrointestinal disorder.
 13. The method of claims 11, wherein the painis chronic pain.
 14. The method of claim 11, wherein the pain isneuropathic pain.
 15. The method of claim 11, further comprisingadministering an opioid drug to the subject.
 16. The method of claim 15,wherein the steps of administering the peptide and the opioid drug areperformed concurrently.
 17. The method of claim 15, wherein the steps ofadministering the peptide and the opioid drug are performed alternatelyon a rotating basis.
 18. The method of claim 11, wherein the step ofadministering the peptide is performed by administering repeated,increasing doses of the peptide to the subject until fullantinociception is achieved, and then maintaining the blood level of thepeptide at the level obtained at full antinociception.
 19. The method ofclaim 11, wherein the peptide is administered parenterally.
 20. Themethod of claim 11, wherein the peptide comprises at least one peptideselected from the group consisting of:Tyr-cyclo[D-Lys-Trp-Phe-Glu]-Gly-NH₂ (SEQ ID NO:3),Tyr-cyclo[D-Glu-Phe-Phe-Lys]-Gly-NH₂ (SEQ ID NO:4), andTyr-cyclo[D-Orn-Phe-p-Cl-Phe-Asp]-Val-NH₂ (SEQ ID NO:7).
 21. A methodfor treating a drug dependence comprising administering to a subject atherapeutically effective amount of the peptide of claim
 1. 22. Themethod of claim 21, wherein the peptide is administered parenterally.23. The method of claim 21, wherein the peptide comprises at least onepeptide selected from the group consisting of:Tyr-cyclo[D-Lys-Trp-Phe-Glu]-Gly-NH₂ (SEQ ID NO:3),Tyr-cyclo[D-Glu-Phe-Phe-Lys]-Gly-NH₂ (SEQ ID NO:4), andTyr-cyclo[D-Orn-Phe-p-Cl-Phe-Asp]-Val-NH₂ (SEQ ID NO:7).
 24. A method ofactivating a mu-opioid receptor comprising contacting the mu-opioidreceptor with the peptide of claim
 1. 25. The method of claim 24,wherein the peptide comprises at least one peptide selected from thegroup consisting of: Tyr-cyclo[D-Lys-Trp-Phe-Glu]-Gly-NH₂ (SEQ ID NO:3),Tyr-cyclo[D-Glu-Phe-Phe-Lys]-Gly-NH₂ (SEQ ID NO:4), andTyr-cyclo[D-Orn-Phe-p-Cl-Phe-Asp]-Val-NH₂ (SEQ ID NO:7).
 26. A methodfor measuring the quantity of a mu opioid receptor in a sample,comprising: (i) contacting a sample suspected of containing a mu opioidreceptor with the peptide of claim 1 to form a compound-receptorcomplex; (ii) detecting the complex formed in step (i); and (iii)quantifying the amount of complex detected in step (ii).
 27. Acompetitive assay method for detecting the presence of a molecule thatbinds to a mu opioid receptor comprising: (i) contacting a samplesuspected of containing a molecule that binds to a mu opioid receptorwith a mu opioid receptor and the peptide of claim 1, wherein thepeptide and receptor form a compound-receptor complex; (ii) measuringthe amount of the complex formed in step (i); and (iii) comparing theamount of complex measured in step (ii) with the amount of a complexformed between the mu opioid receptor and the peptide in the absence ofthe sample.